Team:TU Delft/30 July 2010 content

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=Alkane Sensing, Solvent Tolerance and Salt Tolerance=
+
=Lab work=
 +
 
 +
==Alkane Degradation==
 +
 
 +
====PCR Amplification====
 +
[https://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=29_July_2010 Yesterday's] were ligation mixes were [[Team:TU_Delft/protocols/PCR|amplified]] with the primers PCR F and PCR R. The product was put on [[Team:TU_Delft/protocols/agarose_gel|1% agarose gel]]:
 +
 
 +
Lane Description:
 +
{|style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''Description'''
 +
|'''Expected Length (bp)'''
 +
|'''Primers'''
 +
|'''Status'''
 +
|'''Remarks'''
 +
|-
 +
|1
 +
|PCR product of 007 + 008
 +
|
 +
|PCR F + PCR R
 +
|<font color=limegreen>✓</font>
 +
|
 +
|-
 +
|2
 +
|PCR product of 009 + 010
 +
|
 +
|PCR F + PCR R
 +
|<font color=limegreen>✓</font>
 +
|
 +
|-
 +
|3
 +
|PCR product of 017 + 018
 +
|
 +
|PCR F + PCR R
 +
|<font color=red>✗</font>
 +
|
 +
|-
 +
|4
 +
|PCR product of 019 + B0015
 +
|
 +
|PCR F + PCR R
 +
|<font color=limegreen>✓</font>
 +
|
 +
|-
 +
|5
 +
|PCR product of 007 (negative control)
 +
|
 +
|PCR F + PCR R
 +
|<font color=red>✗</font>
 +
|The primers also anneal without a complete ligation
 +
|-
 +
|M1
 +
|SmartLadder
 +
|n/a
 +
|n/a
 +
|n/a
 +
|
 +
|}
 +
 
 +
====Digestion====
 +
The PCR products were subsequently [[Team:TU_Delft/protocols/restriction_enzyme_digestion|digested]]:
 +
 
 +
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''Sample'''
 +
|'''Enzyme 1'''
 +
|'''Enzyme 2'''
 +
|'''Buffer'''
 +
|'''BSA'''
 +
|'''Needed fragment'''
 +
|-
 +
|1
 +
|40 μL PCR product 007 + 008
 +
|EcoRI 
 +
|PstI
 +
|2 (BioLabs)
 +
|✓
 +
|‘E–007 + 008–P’
 +
|-
 +
|2
 +
|40 μL PCR product 009 + 010
 +
|EcoRI 
 +
|PstI
 +
|2 (BioLabs)
 +
|✓
 +
|‘E–009 + 010-P’
 +
|-
 +
|3
 +
|40 μL PCR product 017 + 018
 +
|EcoRI 
 +
|PstI
 +
|2 (BioLabs)
 +
|✓
 +
|‘E–017 + 018–P’
 +
|-
 +
|4
 +
|40 μL PCR product 019 + B0015
 +
|EcoRI 
 +
|PstI
 +
|2 (BioLabs)
 +
|✓
 +
|‘E–019 + B0015-P’
 +
|-
 +
|5
 +
|1 μg pSB1T3
 +
|EcoRI 
 +
|PstI
 +
|3 (BioLabs)
 +
|✗
 +
|‘E–linear pSB1T3-P’
 +
|-
 +
|5
 +
|1 μg pSB1K3
 +
|EcoRI 
 +
|PstI
 +
|3 (BioLabs)
 +
|✗
 +
|‘E–linear pSB1K3-P’
 +
|}
 +
 
 +
====Ligation====
 +
The digestion products were [[Team:TU_Delft/protocols/ligation|ligated]] at 4 °C:
 +
 
 +
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''BioBrick'''
 +
|'''Fragment 1'''
 +
|'''Fragment 2'''
 +
|'''Final volume'''
 +
|-
 +
|1
 +
|K398012
 +
|1 μL ‘E–009 + 010–P’
 +
|7 μL ‘E–linear pSB1T3-P’
 +
|10 μL
 +
|}
 +
 
 +
==Alkane Sensing, Solvent Tolerance and Salt Tolerance==
''by Pieter''
''by Pieter''
I tried to assemble the remaining biobricks from various sub projects. The goal is to assemble the following:
I tried to assemble the remaining biobricks from various sub projects. The goal is to assemble the following:
-
    * AlkS + E0240
+
* AlkS + E0240
-
    * PalkB + J06702
+
* PalkB + J06702
-
    * J61101 + bbc1
+
* J61101 + bbc1
-
    * J61101 + PhPFDalpha
+
* J61101 + PhPFDalpha
-
    * J61101 + PhPFDbeta  
+
* J61101 + PhPFDbeta  
To achieve this J06702, bbc1, PhPFDalpha and PhPFDbeta were amplified by PCR. The J61101 was cut open with S and P after which the biobricks were inserted. AlkS was inserted in the backbone with E0240.  
To achieve this J06702, bbc1, PhPFDalpha and PhPFDbeta were amplified by PCR. The J61101 was cut open with S and P after which the biobricks were inserted. AlkS was inserted in the backbone with E0240.  
-
==PCR with PFX==
+
====PCR====
-
Per sample the following mix was used:
+
 
-
*1,5 ul 10 mM dNTPs
+
The [[Team:TU_Delft/protocols/PCR|PCR]] protocol was followed for the amplification of the BioBricks.
-
*5 ul Enhancer
+
 
-
*5 ul 10x PFX Buffer
+
PCR reactions
-
*1 ul 50 mM MgSO4
+
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
-
*3 ul 5 pmol/ul Forward Primer
+
|'''#'''
-
*3 ul 5 pmol/ul Reverse Primer
+
|'''Description'''
-
*0.6 ul PFX Polymerase
+
|'''Expected lenght (bp)'''
-
*1 ul DNA Template
+
|'''Primer'''
-
*29.9 ul H2O
+
|'''Status'''
 +
|-
 +
|1
 +
|Marker
 +
|n/a
 +
|n/a
 +
|n/a
 +
|-
 +
|2
 +
|J06702
 +
|869
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|3
 +
|bbc1
 +
|703
 +
|MF + MR2
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|4
 +
|PhPFDalpha
 +
|528
 +
|MF + MR2
 +
|<font color=limegreen>✓</font>
 +
|-
 +
|5
 +
|PhPFDbeta
 +
|430
 +
|MF + MR2
 +
|<font color=limegreen>✓</font>
 +
|}
 +
 
 +
====Digestion====
 +
 
 +
The PCR products were subsequently [[Team:TU_Delft/protocols/restriction_enzyme_digestion|digested]]:
-
PCR Program:
+
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
-
start:
+
|'''#'''
-
*2 min 95 C
+
|'''Sample'''
-
*1 min 50 C
+
|''' Enzyme 1'''
-
*1 min 68 C
+
|'''Enzyme 2'''
 +
|'''Buffer'''
 +
|'''BSA'''
 +
|'''Needed fragment'''
 +
|-
 +
|1
 +
|2 μL AlkS
 +
|EcoRI 
 +
|SpeI
 +
|2 (BioLabs)
 +
|✓
 +
|‘E–AlkS–S’
 +
|-
 +
|2
 +
|3 μL E0240
 +
|EcoRI 
 +
|XbaI
 +
|2 (BioLabs)
 +
|✓
 +
|‘X–E0240–E’
 +
|-
 +
|3
 +
|16 μL PalkB
 +
|SpeI 
 +
|PstI
 +
|2 (BioLabs)
 +
|✓
 +
|‘P–PalkB–S’
 +
|-
 +
|4
 +
|40 μL J06702 PCR product
 +
|XbaI 
 +
|PstI
 +
|2 (BioLabs)
 +
|✓
 +
|‘X–J06702–P’
 +
|-
 +
|5
 +
|6 μL J61101
 +
|SpeI 
 +
|PstI
 +
|2 (BioLabs)
 +
|✓
 +
|‘P–J61101–S’
 +
|-
 +
|6
 +
|40 μL bbc1 PCR product
 +
|XbaI 
 +
|PstI
 +
|2 (BioLabs)
 +
|✓
 +
|‘X–J61101–P’
 +
|-
 +
|7
 +
|40 μL PhPFDalpha PCR product
 +
|XbaI 
 +
|PstI
 +
|2 (BioLabs)
 +
|✓
 +
|‘X–PhPFDalpha–P’
 +
|-
 +
|8
 +
|40 μL PhPFDbeta PCR product
 +
|XbaI 
 +
|PstI
 +
|2 (BioLabs)
 +
|✓
 +
|‘X–PhPFDalpha–P’
 +
|}
-
repeated 25 times:
+
====Ligation====
-
*1 min 95 C
+
The digestion products were [[Team:TU_Delft/protocols/ligation|ligated]] over two nights:
-
*1 min 50 C
+
-
*1 min 68 C
+
-
end:
+
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
-
*10 min 68 C
+
|'''#'''
 +
|'''BioBrick'''
 +
|'''Fragment 1'''
 +
|'''Fragment 2'''
 +
|'''Final volume'''
 +
|-
 +
|1
 +
|AlkS + E0240
 +
|10 μL ‘E–AlkS–S’
 +
|10 μL ‘X–E0240–E’
 +
|25 μL
 +
|-
 +
|2
 +
|J61101 + bbc1
 +
|7 μL ‘P–J61101–S’
 +
|1 μL ‘X–J61101–P’
 +
|10 μL
 +
|-
 +
|3
 +
|J61101 + PhPFDalpha
 +
|7 μL ‘P–J61101–S’
 +
|1 μL ‘X–PhPFDalpha–P’
 +
|10 μL
 +
|-
 +
|4
 +
|J61101 + PhPFDbeta
 +
|7 μL ‘P–J61101–S’
 +
|1 μL ‘X–PhPFDbeta–P’
 +
|10 μL
 +
|}

Latest revision as of 08:22, 7 August 2010

Contents

Lab work

Alkane Degradation

PCR Amplification

Yesterday's were ligation mixes were amplified with the primers PCR F and PCR R. The product was put on 1% agarose gel:

Lane Description:

# Description Expected Length (bp) Primers Status Remarks
1 PCR product of 007 + 008 PCR F + PCR R ✓
2 PCR product of 009 + 010 PCR F + PCR R ✓
3 PCR product of 017 + 018 PCR F + PCR R ✗
4 PCR product of 019 + B0015 PCR F + PCR R ✓
5 PCR product of 007 (negative control) PCR F + PCR R ✗ The primers also anneal without a complete ligation
M1 SmartLadder n/a n/a n/a

Digestion

The PCR products were subsequently digested:

# Sample Enzyme 1 Enzyme 2 Buffer BSA Needed fragment
1 40 μL PCR product 007 + 008 EcoRI PstI 2 (BioLabs) ✓ ‘E–007 + 008–P’
2 40 μL PCR product 009 + 010 EcoRI PstI 2 (BioLabs) ✓ ‘E–009 + 010-P’
3 40 μL PCR product 017 + 018 EcoRI PstI 2 (BioLabs) ✓ ‘E–017 + 018–P’
4 40 μL PCR product 019 + B0015 EcoRI PstI 2 (BioLabs) ✓ ‘E–019 + B0015-P’
5 1 μg pSB1T3 EcoRI PstI 3 (BioLabs) ✗ ‘E–linear pSB1T3-P’
5 1 μg pSB1K3 EcoRI PstI 3 (BioLabs) ✗ ‘E–linear pSB1K3-P’

Ligation

The digestion products were ligated at 4 °C:

# BioBrick Fragment 1 Fragment 2 Final volume
1 K398012 1 μL ‘E–009 + 010–P’ 7 μL ‘E–linear pSB1T3-P’ 10 μL

Alkane Sensing, Solvent Tolerance and Salt Tolerance

by Pieter

I tried to assemble the remaining biobricks from various sub projects. The goal is to assemble the following:

  • AlkS + E0240
  • PalkB + J06702
  • J61101 + bbc1
  • J61101 + PhPFDalpha
  • J61101 + PhPFDbeta

To achieve this J06702, bbc1, PhPFDalpha and PhPFDbeta were amplified by PCR. The J61101 was cut open with S and P after which the biobricks were inserted. AlkS was inserted in the backbone with E0240.

PCR

The PCR protocol was followed for the amplification of the BioBricks.

PCR reactions

# Description Expected lenght (bp) Primer Status
1 Marker n/a n/a n/a
2 J06702 869 G00100 + G00101 ✗
3 bbc1 703 MF + MR2 ✓
4 PhPFDalpha 528 MF + MR2 ✓
5 PhPFDbeta 430 MF + MR2 ✓

Digestion

The PCR products were subsequently digested:

# Sample Enzyme 1 Enzyme 2 Buffer BSA Needed fragment
1 2 μL AlkS EcoRI SpeI 2 (BioLabs) ✓ ‘E–AlkS–S’
2 3 μL E0240 EcoRI XbaI 2 (BioLabs) ✓ ‘X–E0240–E’
3 16 μL PalkB SpeI PstI 2 (BioLabs) ✓ ‘P–PalkB–S’
4 40 μL J06702 PCR product XbaI PstI 2 (BioLabs) ✓ ‘X–J06702–P’
5 6 μL J61101 SpeI PstI 2 (BioLabs) ✓ ‘P–J61101–S’
6 40 μL bbc1 PCR product XbaI PstI 2 (BioLabs) ✓ ‘X–J61101–P’
7 40 μL PhPFDalpha PCR product XbaI PstI 2 (BioLabs) ✓ ‘X–PhPFDalpha–P’
8 40 μL PhPFDbeta PCR product XbaI PstI 2 (BioLabs) ✓ ‘X–PhPFDalpha–P’

Ligation

The digestion products were ligated over two nights:

# BioBrick Fragment 1 Fragment 2 Final volume
1 AlkS + E0240 10 μL ‘E–AlkS–S’ 10 μL ‘X–E0240–E’ 25 μL
2 J61101 + bbc1 7 μL ‘P–J61101–S’ 1 μL ‘X–J61101–P’ 10 μL
3 J61101 + PhPFDalpha 7 μL ‘P–J61101–S’ 1 μL ‘X–PhPFDalpha–P’ 10 μL
4 J61101 + PhPFDbeta 7 μL ‘P–J61101–S’ 1 μL ‘X–PhPFDbeta–P’ 10 μL
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